Regulatory (suppressor) lymphocytes are assumed to play a key role in a number of phenomena, from autoimmunity to transplantation tolerance. However, their mechanism of action remains largely unknown due to the complex nature of the systems studied. To simplify matters, we generated a minimal mouse model of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. We have generated transgenic mice (T/R+) for the genes encoding the alpha and beta chains of the T cell receptor from an encephalomyelitogenic T cell clone. The majority of the T/R+ mice do not develop EAE. In contrast, when T/R+ mice were crossed with RAG-1 KO mice to generate T/R-mice, 100% of the T/R- progeny developed EAE spontaneously. Because the RAG- 1 mutation prevents T/R- mice from generating mature B and T lymphocytes, the only lymphocytes these mice contain are anti-MBP transgenic T-cells. In contrast, T/R+ mice have, in addition to roughly similar numbers of anti-MBP T-cell, some non transgenic (endogenous) alphabeta T-cells with diverse repertoires, as well as gammadelta T-cells.. Thus, T/R- mice constitute a monoclonal system to which defined cellular components can be added and their importance in EAE regulation assessed. Our goal is to understand precisely how this regulation occurs at the cellular and molecular levels. We would like to identify the lymphocyte subpopulation responsible for EAE resistance by crossing T/R+ mice with mice knockout for only one of the lymphoid compartments lacking in T/R- mice, and by injecting purified (sorted) cell subpopulations into T/R-mice before and after the onset of EAE. In addition, we know that the EAE resistance of T/R+ mice is caused by neither a failure in thymic positive selection of MBP specific T cells, nor an increased negative selection of these same cells. Furthermore, anti- MBP T cells in the peripheral lymphoid organs of both types of mice are not anergic. In order to better define the action of regulatory cells on anti-MBP cells, we will also compare the anti- MBP cells of T/R- mice to those of T/R+ mice in the later stages of T cell response, i.e., during "shut off," by activation induced cell death or other anti-inflammatory mechanisms. Finally, evidence for a role of regulatory cells in EAE control is also provided by EAE induction by adoptive transfer of anti- MBP CD4+ TH2 cells. Transfer of MBP specific TH2 cells into TH2 cells into RAG-1 KO or TCR alpha KO mice results in EAE development, however, both normal and TCR delta KO mouse recipients are resistant. This strongly suggests that the presence of alphabeta T cells protects recipients against EAE. We plan to define the requirements of these cells for regulation with regard to the specific cell type, the minimum number of cells necessary for regulation, and the specificity of such cells. The increased understanding of negative regulation and control provided by this defined system is potentially applicable not only to multiple sclerosis, but also to other autoimmune diseases and to the knowledge of tolerance in general.